The murine monoclonal antibody 425 (MAb 425) was raised against the human A431 carcinoma cell line and found to bind to a polypeptide epitope on the external domain of the human epidermal growth factor receptor (EGFR). It was found to inhibit the binding of epidermal growth factor (EGF) at both low and high affinity EGFR sites (Murthy et al., 1987), Enhanced expression of EGFR is found to occur on malignant tissue from a variety of sources thus making MAb 425 a possible agent for the diagnosis and therapeutic treatment of human tumors. Indeed, MAb 425 was found to mediate tumor cytotoxicity in vitro and to suppress tumor cell growth of epidermoid and colorectal carcinoma-derived cell lines in vitro (Rodeck et al., 1987). Radiolabelled MAb 425 has also been shown to bind to xenografts of human malignant gliomas in mice (Takahashi et al., 1987).
EGF is a polypeptide hormone which is mitogenic for epidermal and epithelial cells. When EGF interacts with sensitive cells, it binds to membrane receptors; the receptor EGF complexes cluster and then are internalized in endocytotic vesicles. This is responsible for the phenomenon of "down-regulation". EGF binding induces a tyrosine kinase activity of the receptor molecule and induces synthesis of DNA.
The EGF-receptor is a transmembrane glycoprotein of about 170,000 Daltons (Cohen, 1982). It is the gene product of the c-erb-B proto-oncogene (Downward et al., Nature, Vol. 307, pp. 521-527, 1984). The receptor exists in two kinetic forms: so-called low affinity and high-affinity receptors.
The A431 carcinoma cell line expresses abundant EGF-receptors on its cell surfaces, and thus has been used in many studies to generate anti-EGF-receptor antibodies. However, the receptors on A431 differ from those of other cell types in the carbohydrate moieties attached to the polypeptide. Thus many antibodies raised against A431 membranes are directed against carbohydrates which are not common to all forms of the receptor molecule (e.g. Schreiber, 1983).
Other monoclonal antibodies are reactive with the protein moiety of EGF-receptors. These antibodies display a variety of properties upon binding to EGF-receptors, presumably dependent on the particular portion of the receptor molecule bound, and the isotype of the antibody. Some antibodies mimic some of the effects of EGF (agonists) and some inhibit the effects (antagonists).
Expression of EGF-receptors has been implicated in the progression of tumor growth. The gene for the receptors has been found to be the cellular analogue of the avian viral oncogene v-erb-B (Ulrich, 1984). In addition an association has been detected between late stages of melanoma development and extra copies of the chromosome carrying the receptor gene (Koprowski et al., Somatic Cell and Molecular Genetics, Vol. 11, pp. 297-302, 1985).
Because of EGF-receptors are expressed on a wide variety of solid tumors they provide a suitable target for anti-tumor therapy. However, there is a need in the art for a suitable anti-receptor antibody. Many of the known antibodies have properties which would be deleterious if used as anti-tumor agents. For example, antibodies which mimic the effects of EGF could stimulate the progression of the tumor rather than arresting it. Other antibodies which only bind to high or low affinity receptors could be less than optimally effective because EGF could still exert its effect through the unbound receptors. Still other antibodies convert low affinity receptors to high affinity receptors, which could exacerbate tumor growth rather than inhibiting it. Thus there is a need in the art for an anti-EGF-receptor antibody which would be suitable for anti-tumor therapy.
Although murine MAbs have been used for therapeutic treatment in humans, they have elicited an immune response (Giorgi et al., 1983; Jaffers et al., 1986). To overcome this problem, several groups have tried to "humanize" murine antibodies. This can involve one of two approaches. Firstly, the murine constant region domains for both the light and heavy chain can be replaced with human constant regions. Such "chimeric" murine-human antibodies have been successfully constructed from several murine antibodies directed against human tumor-associated antigens (Sun et al., 1987; Whittle et al., 1987; Liu et al., 1987; Gillies and Wesolowski, 1990). This approach totally conserves the antigen-binding site of the murine antibody, and hence the antigen affinity, while conferring the human isotype and effector functions. In the second approach only the complementarity determining regions (CDRs) from the mouse variable regions are grafted together with human framework regions (FRs) of both the light and heavy chain variable domains (V.sub.L and V.sub.H). It is reasoned that this technique will transfer the critical and major portion of the antigen-binding site to the human antibody (Jones et al., 1986).
CDR grafting has been carried out for several rodent monoclonals (Jones et al., 1986; Reichmann et al., 1988; Verhoeyen et al.; 1988; Queen et al.; 1989; Co et al., 1991; Gorman et al., 1991; Maeda et al., 1991; Temptest et al., 1991). All retained their capacity to bind antigen, although the affinity was usually diminished. In most cases it was deemed necessary to alter certain amino acids in the human framework residues (FRs). Both chimeric and CDR grafted antibodies have proved superior to the mouse antibodies in the clinic (Hale et al., 1988; LoBuglio et al., 1989; Mathieson et al., 1990). However, a general teaching of which amino acids have to be changed, is not known and not completely predictable in any case.
EP 088 994 proposes the construction of recombinant DNA vectors comprising of a DNA sequence which codes for a variable domain of a light or a heavy chain of an immunoglobulin specific for a predetermined ligand. The application does not contemplate variations in the sequence of the variable domain.
EP 102 634 describes the cloning and expression in bacterial host organisms of genes coding for the whole or a part of human IgG heavy chain polypeptide, but does not contemplate variations in the sequence of the polypeptide.
EP 239 400 proposes that humanized antibodies can be obtained by replacing the antigen-binding site (hypervariable regions) of any human antibody by an antigen-binding site of a non-human, for example of a mouse or a rat antibody by genetechnological methods.
Thus, following this teaching, human or humanized antibodies can be manufactured having specific antigen-binding sites which were not available up to now in antibodies originating from humans.
Chimeric antibodies can be obtained by replacing not only the CDRs but the whole variable regions of the light and heavy chains. Chimeric antibodies, however, can still be immunogenic. Chimeric antibodies are, however, very useful for diagnostic purposes and optimizing humanized antibodies.
It could be shown that the affinity of the antigen-binding sites can be influenced by selective exchange of some single amino acids within the variable regions which are not directly part of the CDRs (Reichmann et al., 1988).
As consequence in the worst case, the binding affinity of the antigen can be completely lost if one works according to the teaching of the EP 239 400. This fact could be demonstrated by the inventors of the instant invention, who failed in constructing a correspondingly humanized antibody which was directed to epitopes of the EGF-receptor.
Therefore, it must be considered that the success of such a humanization depends on the constitution and conformation of the used variable regions and their interactions with the corresponding antigen-binding site. Thus, it is not completely predictable whether or which modifications within the variable domains of the antibody are necessary in order to obtain or to improve the binding of the antigen to the antibody.